Interleukin-12 (IL-12), is a cytokine, first described as Natural Killer Stimulatory Factor and Cytotoxic Lymphocyte Maturation factor in the late 1980s (Kobayashi et al., 1989; Stern et al., 1990), is a monocyte/macrophage derived cytokine (D'Andrea et al., 1992; Gazzinelli et al., 1993). IL-12 has become the most promising cytokine for the immunotherapy of many cancers due to its pleiotropic effects both In vitro and in vivo. IL-12 has pronounced mitogenic activity on activated T and Natural Killer (NK) cells via an IL-2 independent mechanism (Gately et al., 1991), it also enances the lytic effects of NK cells (Kobayashi et al., 1989) and can promote the expansion of activated T and NK cells (Gately et al., 1991). However, the major interest in IL-12 for cancer immunotherapy is due to its ability to directly stimulate the production of Interferon-.gamma. (INF-.gamma.) from peripheral blood T and NK cells (Wolf et al., 1991) and its ability to promote the development of Th1 CD4+ T helper cells from naive Th0 cells (Manetti et al., 1993; Scott, 1993). Th1 cells propagate cell mediated immune reactions. Indeed both systemic and intra-tumoural administration of recombinant IL-12 (rIL-12) have shown potent anti-tumour activity in every murine tumour studied and reported to date (Brunda et al., 1993; Nastala et al., 1994). Systemic administration of IL-12 has been shown to elicit potent anti-tumour activity in all murine models studfed to date (Brunda et al., 1993; Nastala et al., 1994). It appears that this anti-tumour effect is mediated via activation of CD8+ T cells and requires INF-.gamma.. Additionally, it Mas been recently shown that rIL-12 is capable of preventing tumour metastasis (Mu et al., 1995) and acute graft versus host disease (Sykes et al., 1995). The most promising results with rIL-12, including total tumour regression and establishment of immunological memory, were obseeed using fibroblasts that had been genetically engineered to secrete IL-12 using an IRES-containing retrovirus. These cells were admixed with tumour cells before being returned to the animal (Tahara et al., 1994). It has been hypothesised that local secretion of cytokines from turhour cells will activate the anti-tumour effectors to establish immunity in vivo by best approximating the natural role of IL-12 in eliciting an immune response.
Although rIL-12 has demonstrated potent anti-tumour effects, all cytokines should be delivered in a manner to reduce toxicity to the patient. Indeed administration of rIL-12 has been shown to give the best therapeutic effects when delivered atlthe site of the tumour (Brunda et al., 1993).
It has been that IL-12 has a synergistic effect with the B7/CD28 interaction between the antigen-presenting cell and the appr riate T cell, in inducing proliferation and promoting a Th1 pattern cytokine production in mitogen activated peripheral blood T cells (Kubin et al., 1994; Murphy et al., 1991).
A major difficulty in the construction of a vector designed to deliver IL-12 results from the way in which IL-12 is naturally sythesised. The functional cytokine is a glycosylated, disulphide linked, heterodimer of molecular weight 70 kDa (p70) which is comprised of two unrelated subunits, a light thain of 35 kDa (p35) and heavy chain of 40 kDa (p40). Functional IL-12 requires co-expression of both of the individual genes which encode each subunit (Gubler et al., 1991),
Using the internal ribosome entry site (IRES) element of the encephalomyocarditis virus to allow cap-independent translation, a retroviral construct that allows coordinated high level expression of IL-12 has been developed (Zitvogel et al., 1994). This construct has subsequently been used to eradicate established murine sarcomas (Tahara et al., 1995). ronzever, the use of IRES elements presents serious problems. To express an additional gene in these constructs, such a B7, would involve an additional IRES element and there would be a risk of homologous recombination between these IRES elements resulting in the splicing out of apportion of the insert.
Another potential drawback of using multiple IRES elements is that it leads to a reduction of expression downstream from each IRES element (Zitvogel et al., 1994) which affects the overall efficiency of any IRES-containing vector where maximal expression is required. Expression from each successive downstream IRES element is lower than from the previous one so the more IRES elements, the lower the expression of the gene products regulated by those located downstream.
Additionally, in the murine model, homodimeric p40 is antagonistic to the activity of heterodimeric p70 (mattner et al., 1993). Thus, where p35 and p40 are translated independently, there is the possibility that some p40 will homodimerise rathkr than heterodimerising with p35. This reduces the pool of p40 available for heterodimerisation, as well as actively inhibiting p70 (i.e. functional IL-12) activity.